A Standardized , Automated Approach for Exosome Isolation and Characterization Using Beckman Coulter Instrumentation

Abstract

INTRODUCTION: Exosomes are small microvesicles, derived from the late endosome, most often described in the literature to be less than 120 nm, released by all cell types, and proven to be involved in cancer metastasis1-3. Exosome characterization and analysis comprise a fast, evolving research area even though their biological function has yet to be completely elucidated. Exosomes contain proteins, lipids, and microRNA capable of regulating an assortment of target genes. Recent studies have suggested that exosomes can serve as biomarkers for future clinical and diagnostic use in not only cancer but many other human diseases4. Exciting new findings have implicated exosomes in cardiovascular diseases5-7, autoimmune syndromes8, and neurodegenerative disorders such as Alzheimer’s9 and Parkinson’s10 disease, in addition to infectious diseases such as tuberculosis11, diphtheria12, and even HIV13. An improved and more efficient isolation and characterization protocol for exosomes and other EVs is critical to advancing this exciting field and experts have recently called for the establishment of standardized methods. EV isolation is particularly tedious, requiring several rounds of differential centrifugation and a density gradient centrifugation step to obtain highly pure vesicles. Downstream challenges involve a standardized method for genetic profiling of encapsulated miRNA. Here, we describe a workflow using automated Biomek methods for centrifugation layering and fractionation, total RNA extraction, and cDNA amplification and clean-up for next generation sequencing. NGS results are reported on benign and cancerous colon cell lines. MATERIALS & METHODS: Preparation of Exosome-Depleted Media: Ultracentrifuged media: 500 mL of standard HI-FBS was added equally to 6 Beckman Coulter Ultra-Clear 94 mL centrifuge tubes (part #: 345777) with an adapter and then placed in a Beckman Coulter Type 45 Ti rotor and spun at 120,000 x g, 18 hours, 4 ̊C in a Beckman Coulter Optima XPN ultracentrifuge. The supernatant of each tube was recovered and aliquoted to 50 mL and stored in the -20 ̊C freezer for future use. 50 mL of the centrifugally-depleted FBS was then added to 450 mL of both MEM and RPMI 1640 media. The media was finally supplemented with 10 mM HEPES and 100 U/mL Penicillin-Streptavidin. Cell Culture: Frozen stocks of HCT 116 (Normal Colon) (ATCC CCL-247) and CCD 841 CoN (Colorectal Carcinoma) (ATCC CRL-1790) lines were thawed and suspended in the separate buffer types and initially added to 6 well culture plates (Becton Dickinson). Cells were expanded as they reached confluency and added to T-175 flasks (Greiner). Briefly, both cell lines were trypsinized, resuspended in appropriate buffer, and centrifuged at 750 x g, 10 min, 20 ̊C in a Beckman Coulter Allegra X-15 R in an SX4750A rotor. Cells were resuspended again in the appropriate buffer and 1 mL was added to vials and placed in the Vi-Cell for analysis. 1 mL of the suspended cells was then added directly to the Vi-Cell and quantified for yield and viability. Differential Centrifugation for Exosome Isolation: Following cell pelleting in the previous step, the cell culture media was filtered through a 0.45 μm filter and centrifuged at 2000 x g for 20 minutes at 4 ̊C. The supernatant was then centrifuged at 10,000 x g for 30 minutes in the Optima XPN ultracentrifuge equipped with a SW 32 Ti rotor to remove cell debris. Again, the supernatant was recovered, filtered through a 0.22 μm membrane, and spun at 100,000 x g A STANDARDIZED, AUTOMATED APPROACH FOR EXOSOME ISOLATION AND CHARACTERIZATION USING BECKMAN COULTER INSTRUMENTATION

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@inproceedings{Schwartz2016AS, title={A Standardized , Automated Approach for Exosome Isolation and Characterization Using Beckman Coulter Instrumentation}, author={Chad Schwartz and Zachary Smith and M . S . Beckman}, year={2016} }